PROJECTSUMMARY/ABSTRACT Glioblastoma (GBM) is a deadly primary brain malignancy with limited therapeutic options. Tumor progression is thought to be driven by stem cell-like cells that evade conventional chemoradiotherapy and anti- angiogenictreatment.Indeed,anti-angiogenictherapyandsubsequentworseningoftumoroxygenationpromote hypoxia-resistant stem cell phenotypes that lead to further tumor progression. However, our understanding of mechanisms that underlie both GBM stem cell (GSC) behavior and its regulation by oxygen tension remains incomplete.InourefforttoidentifynoveltargetablemediatorsoftheGSCphenotype,werecentlydiscoveredthat GPR133(ADGRD1),anorphanmemberoftheadhesionfamilyofGprotein-coupledreceptors,isnecessaryfor initiating tumor growth in vitro and in vivo, both GSC properties, in part by triggering signaling mechanisms that increase cytoplasmic cAMP and lead to transcription of genes necessary for ?stemness?. While GPR133 is absent from normal brain tissue, it is expressed with full penetrance in all GBM specimens tested, regardless of molecularsubtype.Onthebasisofthesefindings,wehypothesizethatGPR133isacriticalcomponentoftumor growthbysupportingtheGSCphenotype.We,therefore,believethatGPR133inhibitionrepresentsanoveland appealingtherapeuticstrategyinGBMthatmeritsfurthertestinganddevelopment. We now seek to expand on our published findings and use patient-derived GBM models to elucidate basic mechanismsofactionofGPR133.Aim1willtestthehypothesesthatGPR133identifiesGBMstemcellsandits knockdown in tumor xenografts slows tumor growth and prolongs survival. Aim 2 will build on our finding that, within each tumor, GPR133 expression is highest in the most hypoxic regions, suggesting regulation by oxygen tension. More specifically, we will determine the effect of intratumoral fluctuations in oxygenation on GPR133 expression by correlating mRNA and protein levels with tumor vascularity and oxygenation using targeted intraoperative biopsies of patient tumors. In addition, Aim 2 will determine whether GPR133 knockdown synergizes with cediranib, an anti-angiogenic agent, to prevent tumor progression after aggravation of tumor hypoxia. Finally, Aim 3 will determine the relative contribution of canonical G protein signaling initiated by GPR133andtransducedbycAMPanditseffectorsRAPandPKA,andadhesionmediatedbyGPR133?slongN- terminalectodomain,tothetranscriptionalregulationofgenesthatsupporttheGSCphenotype. The proposed studies will mechanistically clarify GPR133?s role in tumor progression, including in hypoxia exacerbated by anti-angiogenic therapy. The results of these studies will complement our ongoing small molecule inhibitor and physiological ligand discovery efforts. We envision GPR133 inhibition as a testable novel approachinGBM,eitherbyitselforasapowerful?one-twopunch?whencombinedwithanti-angiogenictherapy, thatcantargetbothhypoxia-vulnerableandhypoxia-resistanttumorcells.